This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have synthesized a number of electrophilic small molecules to interrogate the proteomes of eukaryotic cells. Our compounds incorporate a semi-promiscuous kinase inhibitor scaffold, a terminal alkyne, and a reactive electrophile. This combination targets unique sub-classes of kinases that both bind the scaffold and contain a requisite nucleophile to form a specific covalent bond. Binding is detected using the alkyne and copper-catalyzed "click chemistry" to attach diverse fluorescent and affinity tags. The compounds'cellular targets are identified by mass spectrometry after treating lysates or whole cells then affinity purifying covalently bound proteins. Labeling of proteins in cells or complex mixtures can reveals the physiologically relevant targets of our inhibitors, as well as related molecules. We have also developed a competitive labeling strategy to identify potential non-covalent inhibitors of kinases targeted by our irreversible probes.